(Supported by Human Frontier Science Program Organization (Italy) to F. Ichas) We have found that when mitochondria take up calcium in response to trains of calcium pulses, the cations are sorted and stored on a temporal basis within a confined mitochondrial compartment. This form of calcium storage allows recording of the chronological order of the input signals. When mitochondrial calcium release is triggered, the original temporal sequence of the signals is retrieved in an exact inverse order, cations accumulated first being released last. Our results suggest that spatial organization of calcium within the mitochondrion allows time-resolved retrieval of calcium signals, providing the basis of a mitochondrial memory process. We have extended this study to include the examination of mitochondrial uptake of strontium. Electron-dense granules, approximately 50nm in diamater, form in mitochondria pulsed with calcium or strontium ions, and we proposed that some ions are bound in these granules, while others diffuse freely in the mitochondrial matrix. Using anhydrous processing techniques, plastic sections of various pulsing experiments were prepared for analysis. Energy-dispersive x-ray microanalysis was carried out on the IVEM in STEM mode at 400kV. Over 150 spectra were recorded from 40-50nm square areas in three experiments (mixed Ca/Sr, Ca only, Sr only, and control). We were unable to adequately analyze calcium distribution due to deficiencies in our analysis software, but the strontium experiments showed that strontium is indeed taken up by the granules. There is considerably more strontium in the granules compared to the matrix in mitochondria pulsed with strontium alone. If calcium is pulsed before strontium, the strontium content in the matrix remains the same as that in mitochondria pulsed with strontium alone, but the granules now have the same (low) strontium content as the matrix. After analysis of the sample in which the mitochondria are pulsed first with strontium, then calcium, we should be able to verify the role of compartmentation in "mitochondrial memory". We found no strontium in the intracristal spaces, thus eliminating them as a candidate compartment for sequestering ions. A paper is in preparation, and a presentation was made at the Albany Conference on Frontiers of Mitochondrial Research in September. Ichas, F., De Giorgi, F., Mannella, CA, Marko, M., Colonna, R., Bernardi, P. (1998) A mitochondrial memory of Ca2+ signals: sensing and imaging submitochondrial Ca2+ compartmentation. Albany Conference on Frontiers of Mitochondrial Research.